Members of the phylum Nematoda are numerous and diverse, having adapted to existence in most terrestrial and marine environments. Despite their diverse habitats and life-styles, all nematodes are morphologically and developmentally similar. Some nematode species are free-living; others parasitize a wide variety of plant and animal hosts.
Damage to commercially important crops such as corn and soybeans in the U.S. alone due to plant parasitic nematodes is estimated to be about $5 billion annually. Parasitic nematode species, such as Meloidogyne, feed off plant root tissues and lay eggs that hatch into larvae, which then infest other plants. Nematodes of several species, many of which reproduce parthenogenetically, infect plant roots as sexually uncommitted larvae. Their sexual development is influenced by unknown environmental signals that depend on the health of the plant and the population density of infecting nematodes. If conditions are favorable, the larvae develop into females, which continue to feed, grow, and lay eggs with debilitating effects on the plant; if conditions are unfavorable, the larvae develop into males, which leave the root without causing significant harm. Therefore, damage by the parasite, as well as further reproduction, may be prevented by an agent that induces male development.
Debilitating diseases in domestic animals as well as humans are caused by a variety of nematode parasites that infect and reproduce in the gut, bloodstream, or other tissues. Parasitic nematodes are estimated to affect over a billion people worldwide, primarily in third world countries. These nematodes are almost all sexually reproducing species that require both males and females to be present in the host for reproduction. Therefore, an agent that induces all parasites to develop as males would prevent further reproduction.
Caenorhabditis elegans (C. elegans) is a small, free-living soil nematode found commonly in many parts of the world. It feeds primarily on bacteria and reproduces with a life cycle of about 3 days under optimal conditions. The early embryonic cell divisions include a series of asymmetric asynchronous cleavages in which the germ line acts as a stem-cell lineage, giving rise sequentially to the founder cells (generally five) for the somatic tissue lineages, and finally to the germ line founder cell. Postembryonically, nematodes develop through four larval stages (L1-L4) characterized by different cuticle structures. Among parasitic species, the different larval stages often have evolved to become highly specialized for survival in a particular host or host tissue. Developmentally, some parasite nematodes remain sexually uncommitted until the L4 larval stage.
C. elegans exists as two sexes, a self-fertilizing hermaphrodite and a male. Hermaphrodites have two X (XX) chromosomes, produce both oocytes and sperm, and can reproduce either by self-fertilization or by cross-fertilization with males (XO). A hermaphrodite that has not mated lays about 300 eggs during its reproductive life span. Self-progeny broods consist almost entirely of XX hermaphrodites. Cross-fertilization broods are composed of equal numbers of XX hermaphrodites and XO male cross-progeny. Additionally, XO males arise spontaneously at a low frequency (0.2%) in self-fertilizing hermaphrodite populations as a result of meiotic X-chromosome nondisjunction (Hodgkin et al. (1979) Genetics 91:67-94). Many nematode species related to C. elegans have only true females and males and must reproduce by cross-fertilization. The C. elegans hermaphrodite can be viewed as a modified female that has acquired the ability to produce a small number of sperm in the germ line before switching to oogenesis (Villeneuve and Meyer (1990a) Adv. Genet. 27:117-188).
C. elegans is a relatively simple organism anatomically and genetically. The adult hermaphrodite contains only 959 somatic nucleic, and the adult male only 1031 (Wood (1988) in The Nematode Caenorhabditis elegans, W. B. Wood, ed. (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.), pp. 1-16). The degree of sexual dimorphism, however, is extensive and affects the fates of over 30% of the adult somatic cells. The primary sex determination signal in C. elegans is the ratio of the number of X chromosomes to the number of sets of autosomes: the X:A ratio (Nigon (1951) Biol. Bull. Fr. et Belg. 95:187-225; Madl and Herman (1979) Genetics 93:393-402). Diploid animals with two XX chromosomes (2X/2A=1), triploid animals with three X chromosomes (3X/4A=0.75), and tetraploid animals with four X chromosomes (4X/4A=1), are hermaphrodite. Diploid animals with a single X chromosome (1X/2A=0.5) or tetraploid animals with two X chromosomes (2X/4A=0.5) are male, and triploid animals with two X chromosomes (2X/3A=0.67) are usually male. Thus, a ratio of 0.75 or greater elicits hermaphrodite development, while a ratio of 0.67 or less results in male development.
Ten genes have been identified that are required to assess and transmit the primary signal to the genes responsible for generating sexual dimorphism (for reviews, see Hodgkin (1988) in The Nematode Caenorhabditis elegans, W. B. Wood, ed. (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.), pp. 243-280, and Meyer (1988) Trends Genet. 4:337-342). The ten genes are divided into two classes: (1) X-linked genes that have roles in dosage compensation as well as sex-determination, including three sdc (sex determination and dosage compensation) genes, and the xol-1 (XO lethal) gene, and (2) autosomal genes that are involved only in sex determination, including three tra (transformer) genes, the her-1 (hermaphroditization) gene, and three fem (feminization) genes.
Regulatory genes exhibiting two classes of mutations that result in opposite (or reciprocal) phenotypes have been dubbed "switch" genes because the levels of their products are correlated with one of two alternative developmental decisions (Hodgkin (1984) J. Embryology Exp. Morph. 83: 103-117 (supplement); Sternberg and Horvitz (1984) Ann. Rev. Genet. 18:489-524). Several genes in the C. elegans sex determination pathway are switch genes: tra-1 (Hodgkin (1987) Ann. Rev. Genet. 21:133-154, and (1988) Genes and Develop. 1:731-745), tra-2 (Doniach (1986) Genetics 114:53-76), fem-3 (Barton et al. (1987) Genetics 115:107-119), and her-1 (Hodgkin (1980) Genetics 96:147-164).
The her-1 gene functions at the first step of the sex determination process and is required for male but not for hermaphrodite development (Hodgkin (1980) supra). The gene is defined by over twenty-five recessive loss-of-function (lf) mutations, most of which completely transform XO males into XO self-fertile, anatomically normal hermaphrodites (Hodgkin (1980) supra; Trent et al. (1988) Genetics 120:145-157). Two dominant gain-of-function (gf) her-1 mutations that map to the same locus (n695 and y101) result in the opposite phenotype: XX animals are variably masculinized into pseudo-males; XO animals are unaffected (Trent et al. (1983) Genetics 104:619-647, and (1988) supra). Thus, active her-1 gene product is required for normal male development in XO animals, and its presence is sufficient to induce male differentiation and prevent female development in XX animals.
Hodgkin (1980) supra, has proposed a model in which the her-1, tra, and fem genes regulate sex determination via a hierarchical pathway involving several negative regulatory interactions, shown in FIG. 1. According to this model the X:A ratio sets the activity state (high or low) of the her-1 gene, and the her-1 gene in turn, via the tra-2, tra-3, and fem genes, sets the state of the tra-1 gene, which controls subsequent sexual differentiation. More specifically, her-1 acts as a negative regulator of tra-2 and tra-3, which in turn negatively regulate the fem genes. The fem genes act as negative regulators of tra-1.
This model suggests that in wild-type C. elegans sex determination a variable quantitative primary signal, the X:A ratio, is integrated and transduced into a secondary binary developmental signal: her-1 activity is high (if the X:A ratio is low as in XO animals) or her-1 activity is low (if the X:A ratio is high as in XX animals). How the X:A ratio is assessed and how this signal is relayed to her-1 is not known, although the xol-1, sdc-1, and sdc-2 genes are known to have important roles in this process, and the two sdc genes are known to function as negative regulators of her-1 (Miller et al. (1988) Cell 55:167-183; Villeneuve and Meyer (1987) Cell 48:25-37 and (1990a) supra; Nusbaum and Meyer (1989) Genetics 122:579-593). The regulatory pathway of sex determination in C. elegans (FIG. 1) appears to operate in two different states in the two sexes. In XX hermaphrodites, where the X:A ratio is high, xol-1 activity is low; therefore, sdc- 1 and scd-2 activities are high, and her-1 activity is low. In XO males, where X:A is low, xol-1 activity is high; therefore, sdc-1 and sdc-2 activities are low, and her-1 activity is high. The terminal regulator, tra-1, whose activity is high in XX animals and low in XO animals, controls a variety of downstream sexual differentiation genes (Villeneuve and Meyer (1990a) supra).
This application describes the isolation, sequencing, and expression of the her-1 nucleic acid sequence encoding a protein product that prevents female development and activates the male developmental pathway in nematodes. Further, the her-1 protein has been discovered to be a secretory protein capable of exerting its masculinizing effect on cells exposed to it. This suggests that the her-1 nucleic acid sequence and protein may be useful for the control of parasitic nematodes in plants, animals, and humans.
Although the her-1 gene described herein was isolated from C. elegans, the protein product of the her-1 gene is expected to activate male development in other members of the phylum Nematoda since fundamental sex determining mechanisms are expected to be conserved among different classes of nematodes. Further, the present invention encompasses use of the methods herein described for isolation of the equivalent gene in other species of nematodes, as well as the isolation or synthesis of other molecules which mimic the biological activity of her-1.